Understanding the interplay between dNTP metabolism and genome stability in cancer.

The size and composition of the intracellular DNA precursor pool is integral to the maintenance of genome stability, and this relationship is fundamental to our understanding of cancer. Key aspects of carcinogenesis, including elevated mutation rates and induction of certain types of DNA damage in cancer cells, can be linked to disturbances in deoxynucleoside triphosphate (dNTP) pools. Furthermore, our approaches to treat cancer heavily exploit the metabolic interplay between the DNA and the dNTP pool, with a long-standing example being the use of antimetabolite-based cancer therapies, and this strategy continues to show promise with the development of new targeted therapies. In this Review, we compile the current knowledge on both the causes and consequences of dNTP pool perturbations in cancer cells, together with their impact on genome stability. We outline several outstanding questions remaining in the field, such as the role of dNTP catabolism in genome stability and the consequences of dNTP pool expansion. Importantly, we detail how our mechanistic understanding of these processes can be utilised with the aim of providing better informed treatment options to patients with cancer.

Computational Modeling Study of the Molecular Basis of dNTP Selectivity in Human Terminal Deoxynucleotidyltransferase.

Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme's selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants-containing either substitution D395N or substitutions D395N+E456N-that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications.

The N-terminal SAM domain of the dNTPase SAMHD1 is not required for inhibition by small molecule TH6342.

Sterile alpha motif and histidine-aspartic acid domain containing protein-1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) hydrolase that controls dNTP pools and detoxifies cancer cells of chemotherapy metabolites. TH6342 is a recently reported small molecule inhibitor of SAMHD1 that interacts with the protein in vitro and non-competitively prevents dimerisation, a prerequisite for catalysis. The binding site of TH6342 on SAMHD1 is currently unknown. In the present study we demonstrate that the N-terminal SAM domain of SAMHD1 is not required for inhibition by TH6342.

Substrate Specificity Diversity of Human Terminal Deoxynucleotidyltransferase May Be a Naturally Programmed Feature Facilitating Its Biological Function.

Terminal 2'-deoxynucleotidyl transferase (TdT) is a unique enzyme capable of catalysing template-independent elongation of DNA 3' ends during V(D)J recombination. The mechanism controlling the enzyme's substrate specificity, which is necessary for its biological function, remains unknown. Accordingly, in this work, kinetic and mutational analyses of human TdT were performed and allowed to determine quantitative characteristics of individual stages of the enzyme-substrate interaction, which overall may ensure the enzyme's operation either in the distributive or processive mode of primer extension. It was found that conformational dynamics of TdT play an important role in the formation of the catalytic complex. Meanwhile, the nature of the nitrogenous base significantly affected both the dNTP-binding and catalytic-reaction efficiency. The results indicated that neutralisation of the charge and an increase in the internal volume of the active site caused a substantial increase in the activity of the enzyme and induced a transition to the processive mode in the presence of Mg2+ ions. Surrogate metal ions Co2+ or Mn2+ also may regulate the switching of the enzymatic process to the processive mode. Thus, the totality of individual factors affecting the activity of TdT ensures effective execution of its biological function.

Using Selective Enzymes to Measure Noncanonical DNA Building Blocks: dUTP, 5-Methyl-dCTP, and 5-Hydroxymethyl-dCTP.

Cells maintain a fine-tuned balance of deoxyribonucleoside 5'-triphosphates (dNTPs), a crucial factor in preserving genomic integrity. Any alterations in the nucleotide pool's composition or chemical modifications to nucleotides before their incorporation into DNA can lead to increased mutation frequency and DNA damage. In addition to the chemical modification of canonical dNTPs, the cellular de novo dNTP metabolism pathways also produce noncanonical dNTPs. To keep their levels low and prevent them from incorporating into the DNA, these noncanonical dNTPs are removed from the dNTP pool by sanitizing enzymes. In this study, we introduce innovative protocols for the high-throughput fluorescence-based quantification of dUTP, 5-methyl-dCTP, and 5-hydroxymethyl-dCTP. To distinguish between noncanonical dNTPs and their canonical counterparts, specific enzymes capable of hydrolyzing either the canonical or noncanonical dNTP analogs are employed. This approach provides a more precise understanding of the composition and noncanonical constituents of dNTP pools, facilitating a deeper comprehension of DNA metabolism and repair. It is also crucial for accurately interpreting mutational patterns generated through the next-generation sequencing of biological samples.

The Role of Time Perspective and Mindfulness on Life Satisfaction in the United States of America, Spain, Poland and Japan: A Cross-Cultural Study.

We assessed the cross-cultural role of Time Perspective (TP) tendencies [Past Positive (PP), Past Negative (PN), Present Hedonistic (PH), Present Fatalistic (PF), and Future (F)], the Deviation from a Balanced Time Perspective (DBTP) profile, the Deviation from a Negative Time Perspective (DNTP) profile, and mindfulness on life satisfaction (LS). The sample consisted of psychology undergraduate students (N= 867, MAGE= 20.19, SD= 3.417) in four countries: USA, Spain, Poland and Japan. We used a 17-item short version of the Zimbardo Time Perspective Inventory (ZTPI), the Mindful Attention Awareness Scale (MAAS), and the Satisfaction with Life Scale (SWLS) in all countries. For ensuring measurement invariance, we conducted pairwise CFAs for the ZTPI-17, MAAS and SWLS. Regression analyses showed that PN predicted decreased LS in Poland and Japan. PP predicted increased LS in Spain. F predicted increased LS in Poland. DNTP predicted decreased LS in Poland. Mindfulness predicted decreased LS in Japan and increased LS in USA, Spain and Poland. Moreover, mediation analyses revealed that the DBTP partially mediated the relationship between mindfulness and LS in Spain and USA. The DNTP partially mediated the relationship between mindfulness and LS in Spain, Poland and Japan (opposite direction). The findings suggest that the association of TP, mindfulness and LS differs across the investigated countries as a function of culture.

Analysis of the Arabidopsis venosa4-0 mutant supports the role of VENOSA4 in dNTP metabolism.

Human Sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1) functions as a dNTPase to maintain dNTP pool balance. In eukaryotes, the limiting step in de novo dNTP biosynthesis is catalyzed by RIBONUCLEOTIDE REDUCTASE (RNR). In Arabidopsis, the RNR1 subunit of RNR is encoded by CRINKLED LEAVES 8 (CLS8), and RNR2 by three paralogous genes, including TSO MEANING 'UGLY' IN CHINESE 2 (TSO2). In plants, DIFFERENTIAL DEVELOPMENT OF VASCULAR ASSOCIATED CELLS 1 (DOV1) catalyzes the first step of the de novo biosynthesis of purines. Here, to explore the role of VENOSA4 (VEN4), the most likely Arabidopsis ortholog of human SAMHD1, we studied the ven4-0 point mutation, whose leaf phenotype was stronger than those of its insertional alleles. Structural predictions suggested that the E249L substitution in the mutated VEN4-0 protein rigidifies its 3D structure. The morphological phenotypes of the ven4, cls8, and dov1 single mutants were similar, and those of the ven4 tso2 and ven4 dov1 double mutants were synergistic. The ven4-0 mutant had reduced levels of four amino acids related to dNTP biosynthesis, including glutamine and glycine, which are precursors in the de novo purine biosynthesis. Our results reveal high functional conservation between VEN4 and SAMHD1 in dNTP metabolism.

Hepatitis C Virus Down-Regulates the Expression of Ribonucleotide Reductases to Promote Its Replication.

Ribonucleotide reductases (RRs or RNRs) catalyze the reduction of the OH group on the 2nd carbon of ribose, reducing four ribonucleotides (NTPs) to the corresponding deoxyribonucleotides (dNTPs) to promote DNA synthesis. Large DNA viruses, such as herpesviruses and poxviruses, could benefit their replication through increasing dNTPs via expression of viral RRs. Little is known regarding the relationship between cellular RRs and RNA viruses. Mammalian RRs contain two subunits of ribonucleotide reductase M1 polypeptide (RRM1) and two subunits of ribonucleotide reductase M2 polypeptide (RRM2). In this study, expression of cellular RRMs, including RRM1 and RRM2, is found to be down-regulated in hepatitis C virus (HCV)-infected Huh7.5 cells and Huh7 cells with HCV subgenomic RNAs (HCVr). As expected, the NTP/dNTP ratio is elevated in HCVr cells. Compared with that of the control Huh7 cells with sh-scramble, the NTP/dNTP ratio of the RRM-knockdown cells is elevated. Knockdown of RRM1 or RRM2 increases HCV replication in HCV replicon cells. Moreover, inhibitors to RRMs, including Didox, Trimidox and hydroxyurea, enhance HCV replication. Among various HCV viral proteins, the NS5A and/or NS3/4A proteins suppress the expression of RRMs. When these are taken together, the results suggest that HCV down-regulates the expression of RRMs in cultured cells to promote its replication.

Vpx requires active cellular dNTP biosynthesis to effectively counteract the anti-lentivirus activity of SAMHD1 in macrophages.

HIV-1 replication in primary monocyte-derived macrophages (MDMs) is kinetically restricted at the reverse transcription step due to the low deoxynucleoside triphosphates (dNTP) pools established by host dNTPase, SAM and HD domain containing protein 1 (SAMHD1). Lentiviruses such as HIV-2 and some Simian immunodeficiency virus counteract this restriction using viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates intracellular dNTP pools. However, how dNTP pools increase after Vpx degrades SAMHD1 in nondividing MDMs where no active dNTP biosynthesis is expected to exists remains unclear. In this study, we monitored known dNTP biosynthesis machinery during primary human monocyte differentiation to MDMs and unexpectedly found MDMs actively express dNTP biosynthesis enzymes such as ribonucleotide reductase, thymidine kinase 1, and nucleoside-diphosphate kinase. During differentiation from monocytes the expression levels of several biosynthesis enzymes are upregulated, while there is an increase in inactivating SAMHD1 phosphorylation. Correspondingly, we observed significantly lower levels of dNTPs in monocytes compared to MDMs. Without dNTP biosynthesis availability, Vpx failed to elevate dNTPs in monocytes, despite SAMHD1 degradation. These extremely low monocyte dNTP concentrations, which cannot be elevated by Vpx, impaired HIV-1 reverse transcription in a biochemical simulation. Furthermore, Vpx failed to rescue the transduction efficiency of a HIV-1 GFP vector in monocytes. Collectively, these data suggest that MDMs harbor active dNTP biosynthesis and Vpx requires this dNTP biosynthesis to elevate dNTP levels to effectively counteract SAMHD1 and relieve the kinetic block to HIV-1 reverse transcription in MDMs.

Attenuation of reverse transcriptase facilitates SAMHD1 restriction of HIV-1 in cycling cells.

BACKGROUND: SAMHD1 is a deoxynucleotide triphosphohydrolase that restricts replication of HIV-1 in differentiated leucocytes. HIV-1 is not restricted in cycling cells and it has been proposed that this is due to phosphorylation of SAMHD1 at T592 in these cells inactivating the enzymatic activity. To distinguish between theories for how SAMHD1 restricts HIV-1 in differentiated but not cycling cells, we analysed the effects of substitutions at T592 on restriction and dNTP levels in both cycling and differentiated cells as well as tetramer stability and enzymatic activity in vitro. RESULTS: We first showed that HIV-1 restriction was not due to SAMHD1 nuclease activity. We then characterised a panel of SAMHD1 T592 mutants and divided them into three classes. We found that a subset of mutants lost their ability to restrict HIV-1 in differentiated cells which generally corresponded with a decrease in triphosphohydrolase activity and/or tetramer stability in vitro. Interestingly, no T592 mutants were able to restrict WT HIV-1 in cycling cells, despite not being regulated by phosphorylation and retaining their ability to hydrolyse dNTPs. Lowering dNTP levels by addition of hydroxyurea did not give rise to restriction. Compellingly however, HIV-1 RT mutants with reduced affinity for dNTPs were significantly restricted by wild-type and T592 mutant SAMHD1 in both cycling U937 cells and Jurkat T-cells. Restriction correlated with reverse transcription levels. CONCLUSIONS: Altogether, we found that the amino acid at residue 592 has a strong effect on tetramer formation and, although this is not a simple "on/off" switch, this does correlate with the ability of SAMHD1 to restrict HIV-1 replication in differentiated cells. However, preventing phosphorylation of SAMHD1 and/or lowering dNTP levels by adding hydroxyurea was not enough to restore restriction in cycling cells. Nonetheless, lowering the affinity of HIV-1 RT for dNTPs, showed that restriction is mediated by dNTP levels and we were able to observe for the first time that SAMHD1 is active and capable of inhibiting HIV-1 replication in cycling cells, if the affinity of RT for dNTPs is reduced. This suggests that the very high affinity of HIV-1 RT for dNTPs prevents HIV-1 restriction by SAMHD1 in cycling cells.

Maize LOST SUBSIDIARY CELL encoding a large subunit of ribonucleotide reductase is required for subsidiary cell development and plant growth.

The four-celled stomatal complex consists of a pair of guard cells (GCs) and two subsidiary cells (SCs) in grasses, which supports a fast adjustment of stomatal aperture. The formation and development of SCs are thus important for stomatal functionality. Here, we report a maize lost subsidiary cells (lsc) mutant, with many stomata lacking one or two SCs. The loss of SCs is supposed to have resulted from impeded subsidiary mother cell (SMC) polarization and asymmetrical division. Besides the defect in SCs, the lsc mutant also displays a dwarf morphology and pale and striped newly-grown leaves. LSC encodes a large subunit of ribonucleotide reductase (RNR), an enzyme involved in deoxyribonucleotides (dNTPs) synthesis. Consistently, the concentration of dNTPs and expression of genes involved in DNA replication, cell cycle progression, and SC development were significantly reduced in the lsc mutant compared with the wild-type B73 inbred line. Conversely, overexpression of maize LSC increased dNTP synthesis and promoted plant growth in both maize and Arabidopsis. Our data indicate that LSC regulates dNTP production and is required for SMC polarization, SC differentiation, and growth of maize.

In situ enzymatic template replication on DNA microarrays.

DNA microarrays are very useful tools to study the realm of nucleic acids interactions at high throughput. The conventional approach to microarray synthesis employs phosphoramidite chemistry and yields unmodified DNA generally attached to a surface at the 3' terminus. Having a freely accessible 3'-OH instead of 5'-OH is desirable too, and being able to introduce nucleoside analogs in a combinatorial manner is highly relevant in the context of nucleic acid therapeutics and in aptamer research. Here, we describe an enzymatic approach to the synthesis of high-density DNA microarrays that can also contain chemical modifications. The method uses a standard DNA microarray, to which a DNA primer is covalently bound through photocrosslinking. The extension of the primer with a DNA polymerase yields double-stranded DNA but is also amenable to the incorporation of modified dNTPs. Further processing with T7 exonuclease, which catalyzes the degradation of DNA in a specific (5'→3') direction, results in template strand removal. Overall, the method produces surface-bound natural and non-natural DNA oligonucleotides, is applicable to commercial microarrays and paves the way for the preparation of combinatorial, chemically modified aptamer libraries.

Biocatalytic synthesis of 2'-deoxynucleotide 5'-triphosphates from bacterial genomic DNA: Proof of principle.

2'-deoxynucleoside 5'-triphosphates (dNTPs) are the building blocks of DNA and are key reagents which are incorporated by polymerase enzymes during nucleic acid amplification techniques, such as polymerase chain reaction (PCR). These techniques are of high importance, not only in molecular biology research, but also in molecular diagnostics. dNTPs are generally produced by a bottom-up technique which relies on synthesis or isolation of purified small molecules like deoxynucleosides. However, the disproportionately high cost of dNTPs in low- and middle-income countries (LMICs) and the requirement for cold chain storage during international shipping makes an adequate supply of these molecules challenging. To reduce supply chain dependency and promote domestic manufacturing in LMICs, a unique top-down biocatalytic synthesis method is described to produce dNTPs. Readily available bacterial genomic DNA provides a crude source material to generate dNTPs and is extracted directly from Escherichia coli (step 1). Nuclease enzymes are then used to digest the genomic DNA creating monophosphorylated deoxynucleotides (dNMPs) (step 2). Design and recombinant production and characterization of E. coli nucleotide kinases is presented to further phosphorylate the monophosphorylated products to generate dNTPs (step 3). Direct use of the in-house produced dNTPs in nucleic acid amplification is shown (step 4) and their successful use as reagents in the application of PCR, thereby providing proof of principle for the future development of recombinant nucleases and design of a recombinant solid-state bioreactor for on-demand dNTP production.

Demethylation of dimethyl phosphonate esters with sodium ethanethiolate: Improved synthesis of 5'-methylene substituted 2',5'-deoxyribonucleotides.

We describe a new demethylation method for dimethyl phosphonate esters using sodium ethanethiolate. The new procedure allows demethylation of nucleoside dimethyl phosphonate esters without 1'-α-anomerization, providing an improved synthesis of 5'-methylene substituted 2',5'-deoxynucleotides.

3'dNTP Binding Is Modulated during Primer Synthesis and Translesion by Human PrimPol.

PrimPol is a DNA primase/polymerase from the Archaeo-Eukaryotic Primase (AEP) superfamily that enables the progression of stalled replication forks by synthesizing DNA primers ahead of blocking lesions or abnormal structures in the ssDNA template. PrimPol's active site is formed by three AEP-conserved motifs: A, B and C. Motifs A and C of human PrimPol (HsPrimPol) harbor the catalytic residues (Asp114, Glu116, Asp280) acting as metal ligands, whereas motif B includes highly conserved residues (Lys165, Ser167 and His169), which are postulated to stabilize 3' incoming deoxynucleotides (dNTPs). Additionally, other putative nucleotide ligands are situated close to motif C: Lys297, almost invariant in the whole AEP superfamily, and Lys300, specifically conserved in eukaryotic PrimPols. Here, we demonstrate that His169 is absolutely essential for 3'dNTP binding and, hence, for both primase and polymerase activities of HsPrimPol, whereas Ser167 and Lys297 are crucial for the dimer synthesis initiation step during priming, but dispensable for subsequent dNTP incorporation on growing primers. Conversely, the elimination of Lys165 does not affect the overall primase function; however, it is required for damage avoidance via primer-template realignments. Finally, Lys300 is identified as an extra anchor residue to stabilize the 3' incoming dNTP. Collectively, these results demonstrate that individual ligands modulate the stabilization of 3' incoming dNTPs to optimize DNA primer synthesis efficiency during initiation and primer maturation.

The role of time perspective and acculturative stress on adaptive and maladaptive stress coping strategies of Puerto Ricans living in the island of Puerto Rico and the state of Connecticut in mainland United States.

We assessed the role of Time Perspective (TP) and acculturative stress on adaptive and maladaptive coping strategies, across healthy and treatment-seeking Puerto Ricans living in the island of Puerto Rico (PR), as well as at the state of Connecticut in mainland United States (US). Participants were comprised of 197 adults from the island of PR, as well as 138 adults from Connecticut. TP was measured through five categories assessed by the Zimbardo TP Inventory (Past Positive, Past Negative, Present Fatalistic, Present Hedonistic, and Future), the Deviation from a Balanced Time Perspective-revisited (DBTPr) coefficient, and the Deviation from the Negative Time Perspective (DNTP) coefficient. Acculturative stress was measured with the Acculturative Distress Scale. Adaptive and maladaptive stress-coping were measured through the Brief COPE Inventory. DNTP predicted adaptive coping, whereas acculturative stress, Present Hedonistic, and DBTPr predicted maladaptive coping. Puerto Ricans living in Connecticut engaged more often in maladaptive coping than those in PR. Acculturative stress partially mediated the influence of DBTPr on maladaptive coping. DNTP mediated the influence of state on adaptive coping. DBTPr and acculturative stress totally mediated the influence of state on maladaptive coping. These findings suggest that assessing TP, levels of acculturative stress, and coping strategies could assist in tailoring evidence-based interventions to the specific needs of Puerto Rican populations. Doing so could be effective in promoting a Balanced Time Perspective, reducing acculturative stress, increasing adaptive coping, and improving mental as well as physical health, on Puerto Ricans living in PR or mainland US.

Complex mutation profiles in mismatch repair and ribonucleotide reductase mutants reveal novel repair substrate specificity of MutS homolog (MSH) complexes.

Determining mutation signatures is standard for understanding the etiology of human tumors and informing cancer treatment. Multiple determinants of DNA replication fidelity prevent mutagenesis that leads to carcinogenesis, including the regulation of free deoxyribonucleoside triphosphate pools by ribonucleotide reductase and repair of replication errors by the mismatch repair system. We identified genetic interactions between rnr1 alleles that skew and/or elevate deoxyribonucleoside triphosphate levels and mismatch repair gene deletions. These defects indicate that the rnr1 alleles lead to increased mutation loads that are normally acted upon by mismatch repair. We then utilized a targeted deep-sequencing approach to determine mutational profiles associated with mismatch repair pathway defects. By combining rnr1 and msh mutations to alter and/or increase deoxyribonucleoside triphosphate levels and alter the mutational load, we uncovered previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct the repair of G/C single-base deletions in GC runs, while Msh2-Msh6 specifically directs the repair of substitutions that occur at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds. Finally, we observed that the mutation profiles in double mutants were not necessarily an additive relationship of mutation profiles in single mutants. Our results have implications for interpreting mutation signatures from human tumors, particularly when mismatch repair is defective.

Replication stress triggered by nucleotide pool imbalance drives DNA damage and cGAS-STING pathway activation in NAFLD.

Non-alcoholic steatotic liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. NAFLD has a major effect on the intrinsic proliferative properties of hepatocytes. Here, we investigated the mechanisms underlying the activation of DNA damage response during NAFLD. Proliferating mouse NAFLD hepatocytes harbor replication stress (RS) with an alteration of the replication fork's speed and activation of ATR pathway, which is sufficient to cause DNA breaks. Nucleotide pool imbalance occurring during NAFLD is the key driver of RS. Remarkably, DNA lesions drive cGAS/STING pathway activation, a major component of cells' intrinsic immune response. The translational significance of this study was reiterated by showing that lipid overload in proliferating HepaRG was sufficient to induce RS and nucleotide pool imbalance. Moreover, livers from NAFLD patients displayed nucleotide pathway deregulation and cGAS/STING gene alteration. Altogether, our findings shed light on the mechanisms by which damaged NAFLD hepatocytes might promote disease progression.

Targeting the DNA damage response and repair in cancer through nucleotide metabolism.

The exploitation of the DNA damage response and DNA repair proficiency of cancer cells is an important anticancer strategy. The replication and repair of DNA are dependent upon the supply of deoxynucleoside triphosphate (dNTP) building blocks, which are produced and maintained by nucleotide metabolic pathways. Enzymes within these pathways can be promising targets to selectively induce toxic DNA lesions in cancer cells. These same pathways also activate antimetabolites, an important group of chemotherapies that disrupt both nucleotide and DNA metabolism to induce DNA damage in cancer cells. Thus, dNTP metabolic enzymes can also be targeted to refine the use of these chemotherapeutics, many of which remain standard of care in common cancers. In this review article, we will discuss both these approaches exemplified by the enzymes MTH1, MTHFD2 and SAMHD1. © 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

MYH9 binds to dNTPs via deoxyribose moiety and plays an important role in DNA synthesis.

The accepted notion of dNTP transport following cytoplasmic biosynthesis is 'facilitated diffusion'; however, whether this alone is sufficient for moving dNTPs for DNA synthesis remains an open question. The data presented here show that the MYH9 gene encoded heavy chain of non-muscle myosin IIA binds dNTPs potentially serving as a 'reservoir'. Pull-down assays showed that MYH9 present in the cytoplasmic, mitochondrial and nuclear compartments bind to DNA and this interaction is inhibited by dNTPs and 2-deoxyribose-5-phosphate (dRP) suggesting that MYH9-DNA binding is mediated via pentose sugar recognition. Direct dNTP-MYH9 binding was demonstrated by ELISA and a novel PCR-based method, which showed that all dNTPs bind to MYH9 with varying efficiencies. Cellular thermal shift assays showed that MYH9 thermal stability is enhanced by dNTPs. MYH9 siRNA transfection or treatment with myosin II selective inhibitors ML7 or blebbistatin decreased cell proliferation compared to controls. EdU labeling and cell cycle analysis by flow cytometry confirmed MYH9 siRNA and myosin II inhibitors decreased progression to S-phase with accumulation of cells in G0/G1 phase. Taken together, our data suggest a novel role for MYH9 in dNTP binding and DNA synthesis.